The development of sensitive nonisotopic detection systems for use in biological assays has significantly impacted many research and diagnostic areas, such as DNA sequencing, clinical diagnostic assays, and fundamental cellular and molecular biology protocols. Current nonisotopic detection methods are mainly based on organic reporter molecules that undergo enzyme-linked color changes or are fluorescent, luminescent, or electroactive (Kricka, Ed., Nonisotopic Probing, Blotting, and Sequencing, Academic Press, New York, 1995; Issac, Ed., Protocols for Nucleic Acid Analysis by Nonradioactive Probes, Humana, Totowa, N.J., 1994; and Diamandis and Christopoulos, Eds., Immunoassay, Academic Press, New York, 1996). While these nonisotopic systems solve the problems associated with radioisotopic detection, such as short half-lives of radioisotopes, health hazards and expensive removal of radioactive waste, they are not as sensitive or stable as nonisotopic detection systems that utilize luminescent semiconductor quantum dots. For example, highly luminescent semiconductor quantum dots, such as ZnS-capped CdSe quantum dots, are twenty times brighter, one hundred times more stable against photobleaching, and three times narrower in spectral line width than organic dyes, such as fluorescent rhodamine.
Over the past decade, much progress has been made in the synthesis and characterization of a wide variety of semiconductor quantum dots. Recent advances have led to large-scale preparation of relatively monodisperse quantum dots (Murray et al., JACS 115:8706-15 (1993); Qu et al. (JACS 124:2049 (2002), Nanoletters 1:333 (2001), and Nanoletters 4:465(2004)), Bowen Katari et al., J. Phys. Chem. 98:4109-17 (1994); and Hines et al., J. Phys. Chem. 100:468-71 (1996)). Other advances have led to the characterization of quantum dot lattice structures (Henglein, Chem. Rev. 89:1861-73 (1989); and Weller et al., Chem. Int. Ed. Engl. 32:41-53(1993)) and also to the fabrication of quantum-dot arrays (Murray et al., Science 270:1335-38 (1995); Andres et al., Science 273:1690-93 (1996); Heath et al., J. Phys. Chem. 100:3144-49 (1996); Collier et al., Science 277:1978-81 (1997); Mirkin et al., Nature 382:607-09 (1996); and Alivisatos et al., Nature 382:609-11 (1996)) and light-emitting diodes (Colvin et al., Nature 370:354-57 (1994); and Dabbousi et al., Appl. Phys. Let. 66:1316-18 (1995)). In particular, IIB-VIB semiconductors have been the focus of much attention, leading to the development of a CdSe quantum dot that has an unprecedented degree of monodispersivity and crystalline order (Murray (1993), supra).
Methods of making semiconductor nanocrystals consisting of two elements are well documented in Murray et al. (JACS 115:8706 (1993)), Qu et al. (JACS 124:2049 (2002), Nanoletters 1:333 (2001), and Nanoletters 4:465(2004)), Danek et al. (Materials 8(1): 173 (January 1996)), and Hines and Guyot-Sionnest (J. Phys. Chem. 100:468 (January 1996)). Furthermore, some methods of making alloy semiconductor nanocrystals are also documented, e.g., by Xinhua Zhong et al. (JACS 125:8589 (2003) and JACS 125:12559 (2003)).
The published methods of making nanocrystals require careful timing to stop crystal growth at a desired endpoint. For two-element nanocrystal synthesis, the emission wavelength is determined solely by size and is selected by stopping crystal growth at precise times. For shorter wavelengths (smaller crystals), because of the extremely fast reaction rate, it is often exceptionally difficult to stop the reaction at the right time and/or quickly enough. In addition, the reaction rate is so greatly different from one synthesis batch to the next that the time it takes to reach the endpoint is significantly different and must be adjusted each time crystals are made. Alloy crystal emission wavelength is mostly, though not entirely, determined by current synthesis methods that still require the reaction to be stopped at carefully controlled times. Failure to do so may result in Ostwald ripening, causing a broad emission spectrum. The present invention eliminates the need for careful timing of synthesis endpoints for alloy semiconductor nanocrystals.